Abstract
In this study, two pairs of oligonucleotide primers were designed according to the nucleotide sequence of the internal transcribed spacers (ITSs) of Babesia bigemina and B. bovis isolates from China. The primers were used in a multiplex PCR to detect parasite DNA in blood samples from cattle. There was no cross reactions with B. ovata, B. major, B. sp. Kashi, Theileria annulata, T. sergenti, T. sinensis or normal bovine DNA. The sensitivity of multiplex PCR assay was 1 pg and 10 pg DNA for B. bigemina and B. bovis, respectively. A total of 260 field blood samples collected from cattle in five provinces of China were analyzed by multiplex PCR and light microscopy. PCR testing revealed that 7.3% (19/260) and 5.8% (15/260) of cattle were positive for B. bigemina and B. bovis and 1.2% (3/260) of cattle were co-infected with B. bigemina and B. bovis. Using light microscopy, 2.3% (6/260) and 1.5% (4/260) of cattle were infected by B. bigemina and B. bovis, respectively, and no co-infection was found. The results showed that the multiplex PCR developed in the present study could be an alternative diagnostic tool for the detection of B. bovis and B. bigemina infection in cattle.
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References
Aktas M., Bendele K.G., Altay K., Dumanli N., Tsuji M., Holman P.J. 2007. Sequence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species. Veterinary Parasitology, 147, 221–230. DOI: 10.1016/j.vetpar.2007.04.007.
Alhassan A., Pumidonming W., Okamura M., Hirata H., Battsetseq B., Fujisaki K., Yokoyama N., Igarashi I. 2005. Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood. Veterinary Parasitology, 129, 43–49. DOI: 10.1016/j.vetpar.2004.12.018.
Allred D.R. 2003. Babesiosis: persistence in the face of adversity. Trends in Parasitology, 19, 51–55.
Almeria S., Castellà J., Ferrer D., Ortuño A., Estrada-Peña A., Gutiérrez J.F. 2001. Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection. Veterinary Parasitology, 99, 249–259.
Baneth G., Kenny M.J., Tasker S., Anug Y., Shkap V., Levy A., Shaw S.E. 2004. Infection with a proposed new subspecies of Babesia canis, Babesia canis subsp. present in domestic cat. Journal of Clinical Microbiology, 42, 99–105.
Barros S.L., Madruga C.R., Araujo F.R., Menk C.F., Almeida M.A., Melo E.P.S., Kessler R.H. 2005. Serological survey of Babesia bovis, Babesia bigemina and Anaplasma marginale antibodies in cattle from the semi-arid region of Bahia, Brazil, by enzyme linked immunosorbent assays. Memórias do Instituto Oswaldo Cruz, 100, 513–517. DOI:/S0074-02762005000600003.
Bishop J.P., Adams L.G. 1973. Combination thin and thick blood films for the detection of Babesia parasitemia. American Journal of Veterinary Research, 34, 1213–1214.
Conrad P.A., Kjemtrup A.M., Carreno R.A., Thomford J., Wainwright K., Eberhard M., Quick R., Telford S.R.3rd, Herwaldt B.L. 2006. Description of Babesia duncani n. sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms. International Journal for Parasitology, 36, 779–789. DOI: 10.1016/j.ijpara.2006.03.008.
Figueroa J.V., Chieves L.P., Johnson G.S., Buening G.M. 1993. Multiplex polymerase chain reactionbased assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood. Veterinary Parasitology, 50, 69–81.
Fu Y.B., Lai Z.S. 1997. A report of buffalo babesiosis. Guangxi Journal of Animals Husbandry Veterinary Medicine, 3, 46–48 (In Chinese).
García-Sanmartín J., Nagore D., García-Pérez A.L., Juste R.A., Hurtado A. 2006. Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays. BMC Veterinary Research, 2, 16. DOI: 10.1186/1746-6148-2-16.
Gubbels J.M., de Vos A.P., van der Weide M., Viseras J., Schouls L.M., de Vries E., Jongejan F. 1999. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. Journal of Clinical Microbiology, 37, 1782–1789.
Holman P.J., Bendele K.G., Schoelkopf L., Jones-Witthuhn R.L., Jones S.O. 2003. Ribosomal RNA analysis of Babesia odocoilei isolates from farmed reindeer (Rangifer tarandus tarandus) and elk (Cervus elaphus canadensis) in Wisconsin. Parasitology Research, 91, 378–383. DOI: 10.1007/s00436-003-0984-5.
Kho W.G., Chung J.Y., Sim E.J., Kim M.Y., Kim D.W., Jongwutiwes S., Tanabe K. 2003. A multiplex polymerase chain reaction for the differential diagnosis of Plasmodium falciparum and Plasmodium vivax. Parasitology International, 52, 229–236.
Kim C.M., Blanco L.B.C., Alhassan A., Iseki H., Yokoyama N., Xuan X., Igarashi I. 2008. Development of a Rapid Immunochromatographic Test for Simultaneous Serodiagnosis of Bovine Babesioses caused by Babesia bovis and Babesia bigemina. American Journal of Tropical Medicine and Hygiene, 78, 117–121.
Liu A., Guan G., Du P., Gou H., Liu Z., Liu J., Ma M., Yang J., Li Y., Niu Q., Ren Q., Bai Q., Yin H., Luo J. 2012. Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina. Parasitology International, 61, 658–663. DOI: 10.1016/j.parint.2012.07.004.
Liu J.L., Yin H., Liu G.Y., Guan G.Q., Ma M.L., Liu A.H., Liu Z.J., Li Y.Q., Ren Q.Y., Dang Z.S., Gao J.L., Zhao H.P., Luo J.X. 2008. Discrimination of Babesia major and Babesia ovata based on ITS1-5.8S-ITS2 region sequences of rRNA gene. Parasitology Research, 102, 709–713. DOI: 10.1007/s00436-007-0818-y.
Markoulatos P., Siafakas N., Moncany M. 2002. Multiplex polymerase chain reaction: a practical approach. Journal of Clinical Laboratory Analysis, 16, 47–51.
McCosker P.J. 1981. The global importance of babesiosis. In: Ristic M., Kreier J.P. (Eds.), Babesiosis. Academic Press, New York, NY, 24 pp.
Mclaughlin G.L., Edlind T.D., Ihler G.M. 1986. Detection of Babesia bovis using DNA hybridization. Journal of Protozoology, 33, 125–128.
Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28, E63.
Oliveira M.C.S., Oliveira-Sequeira T.C.G., Regitano L.C.A., Alencar M.M., Néo T.A., Silva A.M., Oliveira H.N. 2008. Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick. Veterinary Parasitology, 155, 281–286. DOI: 10.1016/j.vetpar.2008.04.022.
Passos L.M., Bell-Sakyi L., Brown C.G. 1998. Immunochemical characterization of in vitro culture-derived antigens of Babesia bovis and Babesia bigemina. Veterinary Parasitology, 76, 239–249.
Qin W.X. 2005. Diagnosis and therapy of bovine babesiosis. Today Animal Husbandry and Veterinary Medicine, 8, 10 (in Chinese).
Salih D.A., El Hussein A.M., Seitzer U., Ahmed J.S. 2007. Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State, Southern Sudan. Parasitology Research, 101, 1035–1044. DOI: 10.1007/s00436-007-0583-y.
Taylor M.A., Coop R.L., Wall R.L. 2007. Veterinary Parasitology. Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK, 104–105.
Tenter A.M., Friedhoff K.T. 1986. Serodiagnosis of experimental and natural Babesia equi and B. caballi infections. Veterinary Parasitology, 20, 49–61.
Yang Z.C. 1984. A research concerning a four foci babesiosis outbreak. Veterinary Science in China, 4, 36–37 (in Chinese).
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Liu, J., Guan, G., Liu, A. et al. A PCR method targeting internal transcribed spacers: the simultaneous detection of Babesia bigemina and Babesia bovis in cattle. Acta Parasit. 59, 132–138 (2014). https://doi.org/10.2478/s11686-014-0222-6
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DOI: https://doi.org/10.2478/s11686-014-0222-6